This invention relates to method of preparation and applications of a liquid storage-stable cellular whole blood composition for use with diagnostic test procedures for D-dimer, which is routinely utilized as a biochemical marker to rule out deep vein thrombosis (DVT) and pulmonary embolism (PE).
Venous thromboembolism is a major health concern and occurs in a variety of diseases and causes significant mortality and morbidity. The most common manifestations of venous thromboembolism are deep vein thrombosis (DVT) and pulmonary embolism (PE). DVT is a blood clot (thrombus) that develops in one of the deep veins of the body, usually in the lower leg. It is a serious disorder that can lead to tissue damage, skin lesion, and even loss of the limb. PE is a condition in which a portion of the thrombus breaks loose and lodges in pulmonary arteries. PE may result in sudden death.
D-dimer molecules are the products of fibrin degradation. The presence of D-dimer in plasma at an increased concentration is proof that the fibrinolytic system (breakdown of fibrin clot) is in action in response to coagulation activation. Cutoff points for normal levels of D-dimer in human plasma will vary depending on factors such as gender and age, and may be different for different assay methods. Speaking very generally, therefore, a typical cutoff value for normal levels of D-dimer in human plasma will be from about 200 to about 500 ng/mL. Patients with venous thrombosis (DVT or PE) have significantly higher than normal levels of D-dimer in their blood. If the D-dimer level is significantly above the previously established cutoff value, then imaging procedures such as ultrasound and radiographic methods are performed to confirm that the patient has DVT or PE. Also, other possible causes of elevated D-dimer levels such as cancer, diabetes, trauma, cardiovascular disorders, and hematoma may be investigated. If the D-dimer level is below the cutoff value, then DTV and PE are ruled out, and the patient would not be sent for imaging studies or treated with anticoagulants. Thus, determination of D-dimer levels can be used both as a diagnostic tool and as a cost-savings tool in treatment of patients.
A number of diagnostic tests for D-dimer using different technologies have been described in the literature and introduced to the clinical laboratory market. For example, the Diagnostica Stago STA-LIATEST® D-DI, bioMerieux Vidas® D-Dimer, and Dade Behring Advanced D-Dimer assays are some of the quantitative test methods for determination of D-dimer in plasma. The Diagnostica Stago STA-LIATEST® D-DI is an immuno-turbidimetric assay using microlatex particles to which specific antibodies have been covalently attached. The Vidas® D-Dimer assay uses a two-step enzyme immunoassay sandwich method using enzyme linked fluorescent detection. The Dade Behring Advanced D-Dimer assay is also an immuno-turbidimetric assay. These all involve procedures for testing D-dimer content in plasma.
Unlike the above assays, several assays suitable for point-of-care settings have been introduced to the market for testing D-dimer in whole blood specimens. These, for instance, enable testing to be done at bedside or in a physician's office, with immediate results, rather than having the specimen sent to a laboratory to separate the plasma for testing. Two examples of such rapid qualitative assays for testing D-dimer in whole blood specimens are the SimpliRED® D-dimer assay from Agent Biomedical Ltd. (Australia) and the Clearview Simplify D-dimer assay from American Diagnostica, Inc. The SimpliRED® D-dimer assay is an autologous red cell agglutination assay. The active agent is a chemical conjugate of a monoclonal antibody specific to D-dimer linked to a monoclonal antibody, which binds to the red blood cell surface. The Clearview Simplify D-dimer assay is an immunochromatography test using D-dimer specific murine monoclonal antibody conjugated to colloidal gold particles to detect D-dimer. The antibody-gold-D-dimer complex migrates through a membrane in the aqueous phase until it is captured and concentrated at a zone to which a second D-dimer specific murine antibody has been bound. The concentration of the complexes at this zone causes a pink/purple line to appear on the membrane. In this assay, if D-dimer concentrations are below the clinically established cut-off, no visible line should appear.
Quality control materials are routinely used in clinical diagnostics laboratories to monitor the precision and accuracy of the clinical test methods and procedures, and this is done whether the assay is conducted in the laboratory or at the point of care of patients. For optimal performance, a quality control material should be as sensitive as the actual patient sample to the anticipated analytical variances. Furthermore, the quality control material should be storage-stable, and its analyte target concentrations should challenge the medical decision point of the assay. Other desirable features of a quality control material are low cost, lot-to-lot reproducibility, and ease of manufacturing.
In an early publication describing work on the SimpliRED assay [John et al., Thrombosis Research 58:273 (1990)], group O negative whole blood with D-dimer antigen added in serial dilutions to produce a series of concentrations was used as a control. However, the SimpliRED assay is marketed with plasma-based controls and the control marketed with the Clearview Simplify assay is only a “low molecular weight D-dimer spiking solution”. Additionally, several plasma- and buffer-based D-dimer controls for testing this marker in plasma are currently available in the market. One, the Stago LIATEST® (D-Dimer Control (available from Diagnostica Stago, Asnieres-sur Seine, France) is a bi-level lyophilized control composed of citrated normal and abnormal human plasma for positive and negative levels, respectively. Another, the Dade Behring Advanced D-Dimer Control Plasma 1 and 2 (available from Dade Behring Inc., Deerfield Ill.) is a bi-level lyophilized control comprised of pooled plasma supplemented with a D-dimer preparation, stabilized with Hepes buffer, and preserved with Proclin. Bio-Rad Laboratories (Hercules, Calif.) offers a liquid plasma-based control containing three elevated levels of D-dimer prepared from processed human plasma and preservatives. At this time, however, there is no commercially available storage-stable cellular whole blood D-dimer composition for use as control or calibrator in D-dimer assays intended for whole blood testing.
There exists a need for a storage-stable cellular whole blood based quality control material for use with the D-dimer assays intended for whole blood testing. Such a product would be similar in nature to a patient sample and could be used directly on test materials that are designed for point-of-care testing in that it would not be necessary to separate blood cells from plasma in testing such samples. The present invention satisfies that need and meets the other essential requirements for a quality control material, such as responding the same way to analytical variances as a patient sample by using a combination of human erythrocytes and plasma as the base matrix, having target values that challenge the linear dynamic range of the assay, and providing acceptable opened-vial and closed-vial stabilities for long term use.